The purpose of this research is to understand the molecular mechanisms of virulence of Trypanosoma cruzi during mammalian infection. T. cruzi infection causes Chagas' disease, a leading cause of morbidity and mortality in Latin America. Understanding virulence mechanisms of T. cruzi will allow the rational development of vaccines and new drugs directed against T. cruzi. In contrast to most strains of T. cruzi, an avirulent strain is unable to cause parasitemia in murine infection. We hypothesize the avirulent phenotype is caused by defects in virulence genes and thus, complementation of the avirulent phenotype could be used to identify virulence genes. A cosmid library of virulent strain DNA was electroporated into the avirulent strain parasites, and the parasites injected into a mouse. This mouse developed parasitemia, but a control mouse infected with sham-electroporated parasites did not develop parasitemia. Parasites, obtained from mouse blood, were found to have acquired cosmid DNA and were found to reproducibly cause parasitemia in mice. Thus, the avirulent strain has reverted to virulence and reversion was associated with the acquisition of a cosmid. We hypothesize that the cosmid contained virulence genes that complemented defective genes of the avirulent parasite. This complementation system will be used to identify and characterize virulence genes. There are four parts of this proposal. 1. The cosmid will be recovered from the revertant parasites and retransfected into avirulent parasites to confirm that the cosmid can complement the avirulent phenotype. 2. The gene or genes responsible for complementation of the avirulent phenotype will be identified and sequenced. The gene(s) will be disrupted in the virulent strain to determine whether the strain converts to avirulence, confirming the role of the gene(s) in virulence. 3. The function of the virulence genes will be studied by: sequence comparison to the data bases; comparative studies of mutant and virulent strains; recombinant expression; and immunolocalization. 4. Other avirulent strains will be studied to learn whether there are multiple complementation groups and thus, multiple virulence genes that can be identified and characterized by this approach. The identity and function of the virulence genes can be used to design pharmacologic or vaccine strategies. For instance, surface molecules important for virulence are logical targets for vaccines; enzymatic activities important for virulence are logical targets for pharmaceuticals that block function. Given the inefficacy of the drugs for T. cruzi infection, the lack of a vaccine, and the lack of molecular information about virulence genes, the information generated by the proposed research is needed to develop new strategies against this medically important parasite.